It has been suggested that the X-proline peptide bonds play a key role in the active transport of cations by membrane proteins. In bR, there are eleven proline residues. Three of these (Pro-50, 91 and 186) are deeply embedded in the middle of (x-helices B, C, and F, respectively. These residues are conserved in related rhodopsins derived from several strains of bacteria. The presence of conserved proline residues in stable helices is not generally expected, and diffraction studies indicate that motions occur in these helices, further suggesting an important role for these. The detection by SSNMR of protein backbone changes near X-Pro peptide bonds during the bR photocycle may indicate correlations between the structural changes that are known to occur in the retinal chromophore, and more general protein conformational changes. Information about such correlations will give us a more complete picture of the mechanism of proton pumping and the related structural changes in both the retinal chromophore and protein backbone. [unreadable]In order to examine such backbone changes, we have performed NMR experiments on isotopically labeled bR in the LA, M. and M,,+N states. For doubly labeled [ I- I 3C-X],[ 15N-Pro]-bRs (X=Thr, Tyr, Val), the difference spectroscopy between REDOR and CP/MAS was carried out to assign the 15N chemical shifts of the three helix-embedded prolines and the 13C chemical shifts of the proceeding X amino acids. The changes in these chemical shifts during the bR photocycle reflect the changes in the protein conformation. Good signal-to-noise is mandatory to distinguish individual components in a difference spectrum. Resolution is another crucial factor: it has been indicated that the 15N chemical shift of Pro-91 in the M,, state is shifted downfield by 1.8 ppm relative to that in the light-adapted state.